Development of a Duplex Real-time PCR for Differentiation of Salmonella Typhimurium and Monophasic Serovars

Salmonella Typhimurium is the most Salmonella serovar causing acute gastroenteritis and diarrhea. Serovar 1, 4, [5], 12: i:is considered a monophasic variant of S. Typhimurium that threaten public health. Fifty-eight serologically confirmed Salmonella strains were investigated by PCR using 16S rRNA and fliC genes. All 58 strains harbored 16S rRNA while 21 strains harbored fliC gene that included S. Typhimurium (12), S. Kentucky (6), Salmonella variant strain serotype 1, 4, [5],12:i:(1), S. Lagos (1), and S. Kedougou (1). A duplex TaqMan real-time PCR was performed for differentiating between biphasic S. Typhimurium and monophasic serovar 1, 4, [5], 12:i:using fljB1, 2 and fliB/IS200 in the fliA-fliB intergenic region. Ten out of twelve S. Typhimurium harbored fljB 1, 2, while Salmonella variant strain serotype 1, 4, [5], 12:i:lacked this gene. Thirteen strains (12 S. Typhimurium and the variant strain serotype 1, 4, [5], 12:i:-) were positive for fliB/IS200 that is a specific gene for S. Typhimurium (biphasic and monophasic ). The result of duplex TaqMan real-time PCR indicated that 10 S. Typhimurium strains were biphasic while two S. Typhimurium strains and the variant strain serotype 1, 4, [5], 12:i:lack fljB1,2 and had fliB/IS200 were monophasic S. Typhimurium. It is noticed that prolonged subculture and repeat phase inversion method leads to the formation of flakes that in turn cause wrongly serotype identification, therefore, real-time PCR is rapid and can be used for identifying and differentiating between biphasic and monophasic S. Typhimurium.